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Binding areas of urokinase‐type plasminogen activator–plasminogen activator inhibitor‐1 complex for endocytosis receptors of the low‐density lipoprotein receptor family, determined by site‐directed mutagenesis
Author(s) -
Skeldal Sune,
Larsen Jakob V.,
Pedersen Katrine E.,
Petersen Helle H.,
Egelund Rikke,
Christensen Anni,
Jensen Jan K.,
Gliemann Jørgen,
Andreasen Peter A.
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05511.x
Subject(s) - receptor , plasminogen activator , ldl receptor , serpin , serine protease , biochemistry , chemistry , binding site , urokinase receptor , microbiology and biotechnology , biology , protease , lipoprotein , endocrinology , cholesterol , gene , enzyme
Some endocytosis receptors related to the low‐density lipoprotein receptor, including low‐density lipoprotein receptor‐related protein‐1A, very‐low‐density lipoprotein receptor, and sorting protein‐related receptor, bind protease‐inhibitor complexes, including urokinase‐type plasminogen activator (uPA), plasminogen activator inhibitor‐1 (PAI‐1), and the uPA–PAI‐1 complex. The unique capacity of these receptors for high‐affinity binding of many structurally unrelated ligands renders mapping of receptor‐binding surfaces of serpin and serine protease ligands a special challenge. We have mapped the receptor‐binding area of the uPA–PAI‐1 complex by site‐directed mutagenesis. Substitution of a cluster of basic residues near the 37‐loop and 60‐loop of uPA reduced the receptor‐binding affinity of the uPA–PAI‐1 complex approximately twofold. Deletion of the N‐terminal growth factor domain of uPA reduced the affinity 2–4‐fold, depending on the receptor, and deletion of both the growth factor domain and the kringle reduced the affinity sevenfold. The binding affinity of the uPA–PAI‐1 complex to the receptors was greatly reduced by substitution of basic and hydrophobic residues in α‐helix D and α‐helix E of PAI‐1. The localization of the implicated residues in the 3D structures of uPA and PAI‐1 shows that they form a continuous receptor‐binding area spanning the serpin as well as the A‐chain and the serine protease domain of uPA. Our results suggest that the 10–100‐fold higher affinity of the uPA–PAI‐1 complex compared with the free components depends on the bonus effect of bringing the binding areas on uPA and PAI‐1 together on the same binding entity.